Am. J. Trop. Med. Hyg., 89(6), 2013, pp.

1088–1094
doi:10.4269/ajtmh.13-0041
Copyright © 2013 by The American Society of Tropical Medicine and Hygiene

Detection of Leptospira-Specific Antibodies Using a Recombinant Antigen-Based

Enzyme-Linked Immunosorbent Assay
Hua-Wei Chen, Zhiwen Zhang, Eric S. Halsey, Carolina Guevara, Enrique Canal, Eric Hall, Ryan Maves,
Drake H. Tilley, Tadeusz J. Kochel, and Wei-Mei Ching*
Naval Medical Research Center, Silver Spring, Maryland; Naval Medical Research Unit No.6, Lima, Peru;
Naval Medical Center San Diego, San Diego, California

Abstract. We produced three highly purified recombinant antigens rLipL32, rLipL41, and rLigA-Rep (leptospiral
immunoglobulin-like A repeat region) for the detection of Leptospira-specific antibodies in an enzyme-linked immunosorbent
assay (ELISA). The performance of these recombinant antigens was evaluated using 121 human sera. Among them, 63 sera
were microscopic agglutination test (MAT)-confirmed positive sera from febrile patients in Peru, 22 sera were indigenous
MAT-negative febrile patient sera, and 36 sera were from patients with other febrile diseases from Southeast Asia,
where leptospirosis is also endemic. Combining the results of immunoglobulin M (IgM) and IgG detection from these three
antigens, the overall sensitivity is close to 90% based on the MAT. These results suggest that an ELISA using multiple
recombinant antigens may be used as an alternative method for the detection of Leptospira-specific antibodies.

INTRODUCTION of rapid diagnostics of leptospirosis. Among them, LipL32,

Leptospirosis is caused by spirochaetes of the genus
Leptospira. It is considered to be the most widespread bacte-
rial zoonotic disease in the world and recognized as an emerg-
ing infectious disease.1,2 More than 1.7 million cases of severe
leptospirosis are reported each year, with case mortality rate
about 10%.3 It is most common in developing countries, par-
ticularly in the Caribbean, Latin America, the Indian subcon-
tinent, Southeast Asia, and Oceania, although locally acquired
cases in industrialized countries are well described.1,4–13 In
recent years, a new trend in human leptospirosis outbreaks
related to outdoor recreational activities or natural disasters
has been observed.14–22 Symptoms of leptospirosis are non-
specific and may be easily confused with other febrile ill-
nesses, such as dengue or malaria, which require different
treatment regimens. Therefore, timely diagnosis is essential,
because antibiotic therapy provides the greatest benefit when
initiated early in the course of illness.23,24
Currently, the microscopic agglutination test (MAT) is the
standard serological method for the diagnosis of leptospirosis,
in which whole cell antigens representing different serogroups
of leptospirosis are mixed with serum samples and examined
by dark-field microscopy for agglutination.25 It is technically
complex and time consuming, because the method requires
many serovars of Leptospira to be cultured.26 Culturing
Leptospira to obtain the whole cell antigen is particularly
labor intensive and requires special precautions to prevent
infection of laboratory staff. Because MAT relies on the
detection of antibodies to leptospiral antigens, it is limited by
the low sensitivity when acute serum samples were tested.27
Commercial assays based on Leptospira whole cell antigen
are available in rapid formats amenable for point-of-care use.
However, field evaluations of these assays suggested low sen-
sitivities (39–72%) during the early phase of infection.28–36
Recently, the whole genome sequences of several serovars of
Leptospira were reported.37–40 Hundreds of genes encoding
surface exposed lipoproteins and outer membrane proteins
have now been identified as candidates for the development
*Address correspondence to Wei-Mei Ching, Naval Medical Research
Center, 503 Robert Grant Avenue, Silver Spring, MD 20910. E-mail:
weimei.ching@med.navy.mil
LipL41, and leptospiral immunoglobulin-like A (LigA) pro-
teins may be excellent target antigens for such a rapid diag-
nostic test. All three proteins are present only in pathogenic
Leptospira species and have been shown to be exposed on the
Leptospira cell surface.41–44 LipL32 and LipL41 are also the
most abundant cell surface proteins, and all three proteins are
highly conserved across a broad range of pathogenic lepto-
spiral serovars.42,45,46 Lastly, these three antigens have been
shown to be immunogenic, and they have shown promise as
target antigens for serologic diagnosis.47,48
In this study, we cloned the genes of LipL32, LipL41, and
part of the repeat region in LigA antigens into Escherichia
coli expression vectors. The expressed recombinant proteins
were then purified by affinity chromatography on nickel col-
umn. A panel of 85 human sera from Peru (63 MAT positive
and 22 MAT negative sera) was used to evaluate the potential
of these recombinant antigens to be used as diagnostic
reagents in enzyme-linked immunosorbent assays (ELISAs).
MATERIALS AND METHODS
Bacterial strains and vectors. The genomic DNA of
L. interrogans serovar Copenhageni strain Fiocruz L1-130
(ATCC, Manassas, VA) was used as the template for cloning
of all recombinant proteins. Escherichia coli Top10 (Life Tech-
nologies, Grand Island, NY) was used for general cloning. The
cloned genes were inserted into pET28a (EMD Millipore,
Billerica, MA) for the expression of recombinant proteins in
E. coli BL21 (DE3) (Life Technologies, Grand Island, NY)
under the control of phage T7 lac promoter.49
Recombinant antigen preparation. Cloning of the gene
coding for LipL32, LipL41, and the repeat region of LigA
proteins into the expression vector pET28a. A primer pair
(LipL32f [5¢-GGTGGTCATATGGGTCTGCCAAGCCTA
AAAAGC-3¢] and LipL32r [5¢-CCGCTCGAGCTTAGTCG
CGTCAGAAGCAGC-3¢]) was designed by using the nucle-
otide sequence of the open reading frame for LipL32 from
strain L1-130 (GenBank accession no. AF245281.1). The cod-
ing region of full length protein minus predicted signal peptide
for LipL32 (amino acids 25–272) was amplified by polymerase
chain reaction (PCR) using genomic DNA isolated from L.
interrogans strain L1-130 as the template. The primer pair for

1088
 ELISA FOR THE DETECTION OF LEPTOSPIRA
LipL41 (LipL41f [5¢-GGTGGTCATATGGCTACAGTCGA
TGTAGAATATCC-3¢] and LipL41r [5¢-CCGCTCGAGCTT
TGCGTTGCTTTCATCAACG-3¢]) was designed for the
coding region of full length protein minus predicted signal
peptide for LipL41 (amino acids 22–355), and primer pair for
LigA (LigAf [5¢-AAGAATCATATGGCAGCCTTAGTTT
CTATTTCTGT-3¢] and LigAr [5¢-CGCCTCGAGAATAT
CCGTATTAGAGGAATTCCA-3¢]) was designed for the
coding region of amino acids 312–630. Each PCR product
was digested with NdeI and XhoI and ligated into the expres-
sion vector pET28a. The resulting plasmids contained a
sequence coding His tag at both N and C termini of LipL32,
LipL41, and the repeat region of LigA. Top10 competent cells
were transformed with the ligation mixture, and colonies were
screened for the presence of inserts with the correct size. The
final sequences were confirmed by DNA sequencing of the
resulting plasmid.
Expression and purification of the recombinant LipL32,
LipL41, and LigA proteins. E. coli BL21 (DE3) was trans-
formed with plasmids carrying the LipL32, LipL41, or LigA
insert. The recombinant E. coli colony with high expression
levels of the desired protein was cultured overnight in Over-
night Express Medium TB (EMD Millipore, Billerica, MA) in
the presence of kanamycin (50 mg/L) at 37 °C with shaking at
200 rpm. Cell pellets from 500 mL cultures were resuspended
in 20 mL buffer A of 20 mM Tris·HCl, pH 8.0, and 0.5 M NaCl
after centrifugation. Cells were ruptured by sonication (Ultra-
sonic Liquid Processor Model VirSonic 475; VIRTIS Company,
Gardiner, NY) five times at setting 3 for 10 seconds each time,
with cooling on ice for 1 minute between each sonication. Cell
extract was centrifuged at 10,000 g for 30 minutes at 4 °C in
+
a Thermo centrifuge (model IEC MultiRF; Thermo Scientific,
Waltham, MA). The recombinant LipL32 (rLipL32) and
recombinant LigA (rLigA-Rep) were expressed in soluble
form, but recombinant LipL41 (rLipL41) was expressed as an
inclusion body. For the purification of rLipL32 or rLigA-Rep,
the cell lysate supernatant was applied onto a 3 mL nickel
column (Ni-NTA) equilibrated with 20 mM Tris, pH 8.0,
0.5 M NaCl, and 10 mM imidazole (Hisbind buffer). The
column was washed extensively with 30 mL Hisbind buffer.
The recombinant protein was eluted from the column in a
step gradient of 3 mL of 25, 50, 100, 200, 400, and 600 mM
imidazole in Hisbind buffer. Fractions of 3 mL were collected
and analyzed by sodium dodecyl sulfate - polyacrylamide gel
electrophoresis (SDS-PAGE) for purity. Peak fractions were
pooled and stored at −20 °C until use. For the purification of
rLipL41, the pellets of inclusion body were resuspended in
Hisbind buffer containing 8 M urea by vortexing, placed on a
shaker at room temperature for an additional 10 minutes, and
centrifuged for 30 minutes at 10,000 g. The supernatant was
+
applied onto a 3 mL nickel column (Ni-NTA) equilibrated with
the same buffer containing 8 M urea. The column was washed
extensively with 30 mL Hisbind buffer containing 8 M urea.
The rLipL41 was eluted from the column as described above
for rLipL32 or rLigA-Rep in the presence of 8 M urea. Peak
fractions were pooled for refolding.
Refolding of rLipL41 protein. Refolding of rLipL41 protein
in 8 M urea in Hisbind buffer was achieved by sequential
dialysis with decreasing concentration of urea in buffer A
containing 1 mM (ethylenedinitrilo) tetraacetic acid (EDTA)
and 1 mM dithiothreitol (DTT) and finally, buffer A containing
1 mM EDTA and 1 mM DTT without urea. Before dialysis,
1089
the protein concentration of the eluates from the nickel col-
umn was adjusted to less than 1 mg/mL and dialyzed against
10 volumes of 6 M urea in buffer A for 60 minutes at 4 °C. The
same procedure was repeated with 4, 2, and 1 M urea in buffer
A. The final dialysis was in 10 volumes of the eluates of buffer
A without urea with one initial change of buffer at 60 minutes
and finally, overnight at 4 °C. The refolded rLipL41 was
stored at −20 °C.
ELISA. ELISA was used in the detection of immuno-
globulin M (IgM) and IgG antibodies against rLipL32,
rLipL41, and rLigA-Rep. Different amounts (0.15, 0.3, 0.45,
and 0.6 mg/well) of each antigen were used to coat the ELISA
plate to determine the optimum amount for coating. The
optimal amount was determined to be 0.3 mg/well, because
0.45 and 0.6 mg did not increase the signal. Microtiter plates
(96 well) were coated for 40 hours at 4 °C with recombinant
antigen diluted in phosphate buffered saline (PBS) and blocked
with 10% skim milk in PBS for 1 hour. Patient sera diluted
1:100 in PBS with 5% skim milk were then added to the
plate, incubated for 1 hour at room temperature, and washed
three times for 10 minutes each with 0.1% Tween-20 in PBS.
Peroxidase-conjugated rabbit anti-human IgG (Santa Cruz
Biotechnology, Dallas, TX) at 1:4,000-dilution or anti-
human IgM (Dako, Carpinteria, CA) at 1:1,000 dilution was
added. After 1 hour of incubation at room temperature, the
plates were washed as previously described before the addi-
tion of the 2,2¢-azinobis (3-ethylbenzthiazoline-6-sulfonate)
(ABTS) substrate (Kirkegaard & Perry, Gaithersburg, MD).
Optical density at 405 nm (OD405) was measured after
30 minutes of incubation at room temperature in a plate
reader (Molecular Devices, Sunnyvale, CA).
Human sera. In total, 85 sera collected from individual
febrile patients (63 MAT positive sera with titers greater than
100 against different Leptospira serovars [Bataviae, Bratislava,
Icterohaemorrhagiae, and Varillal] and 22 MAT negative
sera) from the Iquitos area of Peru were received from Naval
Medical Research Unit 6, Lima, Peru. The 22 negative sera
were used as local negative controls to calculate cutoff
values for the ELISA. The sample collection date after onset
of fever is listed in Table 1; 36 archived sera from patients
residing in the leptospirosis-endemic area of Southeast Asia
with other febrile illness (9 patients with scrub typhus,
9 patients with murine typhus, 9 patients with spotted fever-
type rickettsioses, and 9 patients with Q fever) were used as
other control specimens.
Ethics. The study was approved by the Naval Medical
Research Center Institutional Review Board (Case Number
PJT61 and Protocol NMRCD.2000.0006) in compliance with
all applicable federal regulations governing the protection of
human subjects. Informed consent was obtained from all
study participants.
Table 1
Summary of the sample collection date after onset of fever
Group Leptospirosis Local control
Number of sera 63 22
Number of sera with day after onset of fever 60 20
Range (days) 0–44 0–71
Median (days) 16 5
Number of sera between 0 and 7 days 22 14
Number of sera between 8 and 20 days 17 2
Number of sera > 21 days 21 4
1090 CHEN AND OTHERS

RESULTS

Production of rLipL32, rLipL41, and rLigA-Rep. LipL32

and LipL41 are highly conserved immunodominant cell sur-
face proteins among pathogenic leptospiral serovars. The Lig
protein family has three members, LigA, -B, and -C, all with
high molecular mass.44,47,50 These Lig proteins have 12–13 tan-
dem bacterial Ig-like repeat domains. The first six domains
from the amino terminus are highly conserved among different
strains.46 The coding region of amino acids 312–630 of LigA
consists of repeat domains three, four, five, and part of six. It is
98.5% homologous to the same domains from LigB within the
same strain. The percentage of DNA sequence identity of LigB
among different strains of leptospira ranges from 67.9% to
97.1%.46 The rLipL32 and rLigA-Rep were purified under the
native condition, and the rLipL41 was purified under the dena-
tured condition. Fractions collected during different steps of
the purification procedure were analyzed by SDS-PAGE
(Figure 1A). The rLipL32, rLipL41, and rLigA-Rep were
highly purified using nickel column (Figure 1B).
IgM and IgG ELISA results for patient sera. The purified
rLipL32 and rLigA-Rep and refolded rLipL41 were used as
antigens to detect the presence of IgM and IgG specific for
Leptospira in an ELISA. The IgM ELISA results for 63
leptospirosis serum samples against rLipL32, rLipL41, and
rLigA-Rep are shown in Figure 2A–C, respectively, and the
IgG ELISA results for 63 leptospirosis serum samples against
rLipL32, rLipL41, and rLigA-Rep are shown in Figure 3A–C,

Figure 2. IgM ELISA results for 63 MAT-positive patient sam-

ples using recombinant proteins. Specific IgM against (A) rLipL32,
(B) rLipL41, and (C) rLigA-Rep were tested. The x axis indicates the
number of days after the onset of illness, and the y axis indicates the
OD405. For three samples missing the days after onset of fever infor-
mation, day 16 (medium day) was used to plot. The cutoff values were
0.189 for rLipL32, 0.180 for rLipL41, and 0.142 for rLigA-Rep
(mean of 22 negative controls plus 2.3 SDs for 95% confidence level).

respectively. Table 2 lists the results of patient samples that

Figure 1. Purification and characterization of rLipL32, rLipL41,
and rLigA-Rep. SDS-PAGE profile for the purification of rLigA-
Rep as an example for recombinant protein expressed in BL21 (DE3).
The fractions of the nickel column affinity chromatography were ana-
lyzed by SDS-PAGE. Lane 1, molecular mass marker; lane 2, starting
material before loaded onto the column; lane 3, fraction of wash; lanes
4–9, fractions of the eluate with 25, 50, 100, 200, 400, and 600 mM
imidazole, respectively. (B) Purified recombinant proteins. Lane 1, molec-
ular mass marker; lane 2, rLipL32; lane 3, rLipL41; lane 4, rLigA-Rep.
had specific IgM and IgG antibodies against each recombi-
nant antigen. There were 7 (11%) and 39 (62%) samples that
showed detectable IgM and IgG against rLipL32, respec-
tively; 8 (13%) and 33 (52%) samples that showed detectable
IgM and IgG against rLipL41, respectively; and 15 (24%) and
33 (52%) samples that showed detectable IgM and IgG
against rLigA-Rep, respectively. Patient samples that had
specific IgM and IgG against any one of these antigens were
24 (38%) and 50 (79%), respectively.
Combining IgM and IgG positive specimens, the sensitivity
of the ELISA using one single antigen ranged from 62% to
65% (Table 3) (rLipL32, 65%; rLipL41, 62%; rLigA-Rep,
63%). The samples that had specific antibodies (IgG or IgM)
from different combinations of either rLipL32 or rLipL41,
rLipL32 or rLigA-Rep, rLipL41 or rLigA-Rep, and rLipL32,
 ELISA FOR THE DETECTION OF LEPTOSPIRA 1091

tion (Table 1). There is a wide range of collection date for the
MAT negative local controls, but more than one-half of them
were collected in the first 7 days after onset of fever (Table 1).
The MAT-positive samples were collected evenly throughout
the 44 day period. The IgM responses were only detected in
five (23%) samples among those samples collected in the first
7 days after onset of fever. The percentage increased to 53%
among the samples collected between days 8 and 20 and
dropped back to 43% for the samples collected between 21
and 44 days after onset of fever (Table 4). The IgG responses
were detected in 68%, 71%, and 95% samples among those
samples collected during days 0–7, 8–20, and 21–44 after
onset of fever, respectively (Figures 2 and 3 and Table 4).
In 63 MAT-confirmed patient sera used in this study, the
sera with the highest agglutinating titers against the serovars
Bataviae, Bratislava, Icterohaemorrhagiae, and Varillal were
10, 29, 12, and 12, respectively (Table 5). Among 10 patient
sera with the highest serovar Bataviae titers, more samples
had IgG antibodies to rLipL32 than rLipL41 or rLigA-Rep.
Among 29 patient sera with the highest serovar Bratislava
titers, the rLigA-Rep IgG ELISA had the highest number of
positive followed by rLipL32 and rLipL41. Among 12 patient
sera with the highest serovar Icterohaemorrhagiae titers,
75% of sera had IgG antibodies to rLipL32 or rLigA-Rep,
and 58% of sera had IgG antibodies to rLipL41 above the
cutoff value. Among 12 patient sera with the highest serovar
Varillal titers, 67% of sera had IgG antibodies to rLipL32 or
rLipL41, and 33% of sera had IgG antibodies to rLigA-Rep
above cutoff value.

Figure 3. IgG ELISA results for 63 MAT-positive patient sam-
ples using recombinant proteins. Specific IgG against (A) rLipL32,
(B) rLipL41, and (C) rLigA-Rep were tested. The x axis indicates the
number of days after the onset of illness, and the y axis indicates the
OD405. For three samples missing the days after onset of fever infor-
mation, day 16 (medium day) was used to plot. The cutoff values were
0.199 for rLipL32, 0.330 for rLipL41, and 0.207 for rLigA-Rep
(mean of 22 negative controls plus 2.3 SDs for 95% confidence level).
or rLipL41, or rLigA-Rep were 53 (84%), 52 (83%), 45 (71%),
and 57 (90%), respectively (Table 3). Of 63 MAT positive
sera, 60 sera have the known collection date after onset of
fever, and of 22 local controls, 20 controls have that informa-
DISCUSSION
The diagnosis of leptospirosis relies mainly on serological
methods. The current serologic gold standard, MAT, is com-
plex and time consuming. The performance of MAT requires
knowledge of the prevalent Leptospira strains in a particular
region and the maintenance of a large panel of leptospiral
cultures.51 The agglutination of Leptospira is thought to be
mediated by lipopolysaccharide (LPS)-specific total anti-
bodies, including both IgM and IgG.52 The LPS varies among
different serovars with low cross-reactivity; therefore, a large
number of different serovars need to be cultured and main-
tained for agglutination tests, such as MAT. Leptospiral pro-
teins with a high degree of sequence homology among various
serovars have a potential advantage compared with serovar-
specific LPS antigens. LipL32 and LigA each exhibits 99%
amino acid sequence homology across a broad range of patho-
genic Leptospira species, whereas a 95% homology has been
shown for LipL41.42,46 Recombinant proteins are much easier
to prepare and standardize, which lead to better batch to batch

Table 2
Summary of ELISA results for IgM and IgG responses against rLipL32, rLipL41, and rLigA-Rep in patient sera
No. (%) positive against

IgM IgG

Group No. of sera L32* L4* Lig* Com† L32* L41* Lig* Com†

Leptospirosis 63 7 (11) 8 (13) 15 (24) 24 (38) 39 (62) 33 (52) 33 (52) 50 (79)

Local control 22 1 (5) 0 (0) 1 (5) 2 (9) 1 (5) 0 (0) 1 (5) 2 (9)
Other control‡ 36 0 (0) 0 (0) 0 (0) 0 (0) 0 (0) 0 (0) 0 (0) 0 (0)
*L32, L41, and Lig represent rLipL32, rLipL41, and rLigA-Rep, respectively.
†Sera that have antibody levels above cutoff values against at least one antigen.
‡Sera from patients with other febrile illness (nine patients with scrub typhus, nine patients with murine typhus, nine patients with spotted fever-type rickettsioses, and nine patients with Q fever).
1092 CHEN AND OTHERS

Table 3
Analysis of different combinations of ELISA data using rLipL32, rLipL41, and rLigA-Rep as the antigen (have detectable IgM or IgG)
No. (%) positive against

Group No. of sera L32* L41* Lig* L32 + L41* L32 + Lig* L41 + Lig* L32 + L41 + Lig*

Leptospirosis 63 41 (65) 39 (62) 40 (63) 53 (84) 52 (83) 45 (71) 57 (90)

Local control 22 2 (9) 0 (0) 2 (9) 2 (9) 4 (18) 2 (9) 4 (18)
*L32, L41, and Lig represent rLipL32, rLipL41, and rLigA-Rep, respectively.

consistency compared with leptospiral LPS preparation using
culture based methods.
In this study, we produced three recombinant immunogenic
antigens, rLipL32, rLipL41, and rLigA-Rep, to explore the
feasibility of using them for the detection of Leptospira-
specific antibodies in ELISA. All three proteins showed sim-
ilar overall sensitivity (62–65%) (Table 3) and high specificity
individually (greater than 90%) (Table 3). Not every serum
had detectable IgM or IgG against these recombinant anti-
gens. Among 63 positive sera, 4, 4, and 11 only had IgM
against rLipL32, rLipL41, and rLigA-Rep, respectively, and
10, 3, and 2 only had IgG against rLipL32, rLipL41, and
rLigA-Rep, respectively. Of 50 IgG-positive samples, 17 of
them were also IgM positive. There were 8, 2, and 1 patient
sera that had specific antibodies against only rLipL32,
rLipL41, and rLigA-Rep, respectively.
Guerreiro and others52 previously showed, using IgG
immunoblotting with whole cell antigens, that LipL32 was
recognized by 37% and 84% of acute and convalescent phase
sera, respectively, and that LipL41 was recognized by 21%
and 36% of acute and convalescent phase sera, respectively.
Similar findings were also reported by Flannery and others53
using recombinant antigens LipL32 and LipL41 in IgG
ELISA.53 The recombinant LipL32 was recognized by 56% and
94% of acute and convalescent phase sera, and LipL41 was
recognized by 24% and 44% of acute and convalescent phase
sera. In both studies, the majority of the patient samples
had the highest agglutinating titers to reference strains of
the serovar Icterohaemorrhagiae. Table 4 showed that
rLipL32 was recognized by 55% and 71% of days 0–7 and
21–44 sera, respectively, and that rLipL41 was recognized by
45% and 67% of days 0–7 and 21–44 sera, respectively. Our
data indicated that, even with highly conserved proteins (such
as LipL32 and LipL41), one antigen was unable to detect
antibodies in all sera from different serovar infections. Our
recombinant proteins have been derived from a strain of
L. interrogans. Therefore, the test results were reasonably
good, because the majority of this panel consists of infections
with serovars that belong to L. interrogans. However, data
from 15 Thai samples showed that these three recombinant
antigens could also detect antibodies in samples with the
highest agglutinating titers against other serovars, such as
Cynopteri (L. kirschneri), Panama (L. kirschneri), and Sejroe
(L. borgpetersenii) (Chen H-W and others, unpublished results).
This result strongly suggests the broadness of the assay using
the combination of conserved antigens.
All three antigens showed robust IgG and weak IgM inter-
actions to the sera of infected patients. They could be the
results of previous exposures to Leptospira infections, which
is quite common in a highly endemic area. Several studies
have documented IgG immunoblot reactivity to leptospiral
proteins during acute-phase illness in the absence of specific
IgM.52–55 The predominant humoral responses during acute-
phase infection were thought to be IgM antibodies directed
primarily against carbohydrate epitopes.56,57 These findings
suggest that early host immune response to Leptospira infec-
tion is characterized by both IgM and IgG specific for differ-
ent moieties, similar to processes observed in Borrelia and
Treponema infections.58–60 As shown in Table 4, more sam-
ples had detectable IgG than IgM in all three stages. There
was no indication of early detection of IgM in the samples that
we evaluated. The percentage of samples that had detectable
IgG against recombinant antigens increased from early (68%;
days 0–7) to late stage (95%; days 21–44). The purpose of the
original study for febrile patients was to determine the sero-
prevalence of different diseases, and therefore, the outcomes
from these leptospirosis patients were not followed.
The combination of results from rLipL32 and rLipL41 only
gave a sensitivity of 84% and a specificity of 91% (Table 3).
The combination of ELISA results from rLipL32, rLipL41,
and rLigA-Rep showed an increase in the assay sensitivity to
90%, whereas the specificity dropped to 82% in the local con-
trol (Table 3). The low specificity is the cumulative non-specific
interactions of rLipL32 and rLigA-Rep. No false positive was
found among the local control against rLipL41 in ELISA. Both
rLipL32 and rLigA-Rep had two false positives among the
local control. To further improve the performance of the
ELISA, we plan to include additional immunogens (such as
LipL21 and OmpL1) to increase the assay’s sensitivity. The
cross-reactive epitopes, which cause false positives, may be

Table 4
Antibody responses against rLipL32, rLipL41, and rLigA-Rep according to days after onset of fever in leptospirosis patient sera (only 60 of
63 leptospirosis patient sera have the information for days after onset of fever)
No. (%) positive against

IgM IgG
Range after onset
of fever (days) No. of sera L32* L4* Lig* Com† L32* L41* Lig* Com† M + G‡

0–7 22 1 (5) 2 (9) 2 (9) 5 (23) 12 (55) 10 (45) 12 (55) 15 (68) 18 (82)
8–20 17 2 (12) 4 (24) 7 (41) 9 (53) 9 (53) 7 (41) 7 (41) 12 (71) 16 (94)
21–44 21 4 (19) 2 (10) 5 (24) 9 (43) 15 (71) 14 (67) 12 (57) 20 (95) 20 (95)
*L32, L41, and Lig represent rLipL32, rLipL41, and rLigA-Rep, respectively.
†Sera that have antibody levels above cutoff values against at least one antigen.
‡Have detectable IgM or IgG.
 ELISA FOR THE DETECTION OF LEPTOSPIRA
Table 5
Comparison of the IgG responses against rLipL32, rLipL41, and
rLigA-Rep in 63 leptospirosis patient sera with primary infecting
serovar by ELISA
No. (%) positive against
Serovar* No. of sera rLipL32 rLipL41 rLigA-Rep
Bataviae 10 7 (70) 4 (40) 4 (40)
Bratislava 29 15 (52) 14 (48) 16 (55)
Icterohaemorrhagiae 12 9 (75) 7 (58) 9 (75)
Varillal 12 8 (67) 8 (67) 4 (33)
*If the sample reacts with more than one serovar by MAT, the serovar with the highest
agglutinating titers is listed.
†Sera that have IgG against at least one specific antigen.
identified by pepscan and subsequently truncated from LipL32
or LigA to improve the assay’s specificity. This study is a pilot
study to show the potential of using multiple recombinant anti-
gens for the detection of Leptospira-specific antibodies. In
addition, a larger number of samples with the information of
the collection date after onset of fever and paired acute and
convalescent phase samples will be needed for future study.
Received January 22, 2013. Accepted for publication September 14, 2013.
Published online October 28, 2013.
Acknowledgments: We are grateful to all the participants in the study
and all the medical and administrative staff who assisted in the collec-
tion of patient samples.
Financial support: This research was supported by the Naval Medical
Research Center (research work unit 6000.RAD1.J.A0310) and the
US Department of Defense Global Emerging Infections Systems
Research Program (work unit number: 847705.82000.25GB.B0016).
Disclaimer: The views expressed in this article are the views of the
authors and do not necessarily reflect the official policy or position of
the Department of the Navy, the Department of Defense, or the US
Government. E.S.H., C.G., E.C., E.H., R.M., D.H.T., T.J.K., and
W.-M.C. are employees of the US Government. This work was pre-
pared as part of their official duties. Title 17 U.S.C. §105 provides
that “Copyright protection under this title is not available for any
work of the United States Government.”
Authors’ addresses: Hua-Wei Chen, Zhiwen Zhang, Eric Hall, Tadeusz
J. Kochel, and Wei-Mei Ching, Naval Medical Research Center, Silver
Spring, MD, E-mails: Hua-Wei.Chen@med.navy.mil, Zhiwen.Zhang@
med.navy.mil, Eric.Hall@med.navy.mil, Tad.Kochel@med.navy.mil,
and WeiMei.Ching@med.navy.mil. Eric S. Halsey, Carolina Guevara,
Enrique Canal, and Drake H. Tilley, Naval Medical Research Unit
No. 6, Lima, Peru, E-mails: Eric.Halsey@med.navy.mil, Carolina.
Guevara@med.navy.mil, Enrique.Canal@med.navy.mil, and Drake
.Tilley@med.navy.mil. Ryan Maves, Naval Medical Center San Diego,
San Diego, CA, E-mail: Ryan.Maves@med.navy.mil.
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